Down-Regulation of the INK4 Family of Cyclin-Dependent Kinase Inhibitors by Tax Protein of HTLV-1 Through Two Distinct Mechanisms

Virology 259, 384–391 (1999) Article ID viro.1999.9760, available online at http://www.idealibrary.com on

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provided by Elsevier - Publisher Connector Down-regulation of the INK4 Family of Cyclin-Dependent Kinase Inhibitors by Tax Protein of HTLV-1 through Two Distinct Mechanisms

Takeshi Suzuki, Tomoko Narita, Masami Uchida-Toita, and Mitsuaki Yoshida1

Department of Cellular and Molecular Biology, Institute of Medical Science, The University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan Received January 18, 1999; returned to author for revision March 8, 1999; accepted April 9, 1999

Tax oncoprotein of human T-cell leukemia virus type 1 (HTLV-1) affects multiple regulatory processes of infected cells through activation and repression of specific transcription and also through modulation of functions of cell cycle regulators. Previously, we found that Tax binds to p16ink4a, a member of the INK4 family of cyclin-dependent kinase inhibitors, and counteracts its inhibitory activity, resulting in cell cycle progression. In this study, we examined the effects of Tax on other members of the INK4 family and found that Tax can bind to p15ink4b similarly to p16ink4a, but not to p18ink4c and p19ink4d. Tax binding to p15ink4b inactivated its function and restored CDK4 kinase activity. Accordingly, Tax-expressing cells became resistant to p15ink4b-mediated growth arrest induced by TGF␤. On the other hand, expression of p18ink4c was transcrip- tionally repressed by Tax through the E-box element of the promoter, which may contribute to the marked reduction of p18ink4c mRNA in HTLV-1-infected T-cells. These observations indicate that Tax suppresses the inhibitory activities of INK4 family members through two independent mechanisms: functional inhibition of two INK4 proteins and repression of expression of another INK4 protein. These effects may play roles in HTLV-1-induced deregulation of the cell cycle, possibly promoting cellular transformation. © 1999 Academic Press

INTRODUCTION Reddy, 1995; Hunter and Pines, 1994). Tax protein of HTLV-1 binds to one of the INK4 family members, Human T-cell leukemia virus type 1 (HTLV-1) (Poeisz et p16ink4a, and suppresses its inhibitory activity, causing al., 1980; Yoshida et al., 1982) is the causative agent of a activation of CDK4. Tax consequently rescued cells from unique T-cell malignancy, adult T-cell leukemia (ATL) G1 arrest, which was induced by overexpression of (Hinuma et al., 1981; Uchiyama et al., 1977). The virally p16ink4a, and resulted in cell proliferation (Suzuki et al., encoded Tax protein is required for viral replication and 1996). Thus, these results provided a novel insight into has been suggested to contribute to the oncogenesis the abnormal regulation induced by HTLV-1 infection. associated with HTLV-1 infection (Yoshida, 1993). Tax The INK4 family consists of p16ink4a (Serrano et al., was originally identified as a transcriptional activator on 1993), p15ink4b (Hannon and Beach, 1994), p18ink4c, the one hand and as a repressor on the other hand, and p19ink4d (Chan et al., 1995; Guan et al., 1994; Hirai affecting specific cellular gene expression (Brauweiler et al., 1997; Inoue et al., 1986; Jeang et al., 1990; Lemasson et al., 1995), all of which have ankyrin motifs. Since Tax et al., 1997; Yoshida, 1995). In addition to transcriptional can bind to the ankyrin motifs of p16ink4a, we examined regulation, Tax was found to inactivate a cyclin-depen- the effects of Tax on INK4-mediated regulation. In this dent kinase (CDK) inhibitor, p16ink4a protein, and dereg- paper, we describe two independent mechanisms by ulate cell cycle control (Suzuki et al., 1996). which Tax inhibited the regulation controlled by the INK4 Cell cycle progression in eukaryotes is controlled by family: direct binding to p15ink4b and transcriptional sequential activation and subsequent inactivation of a repression of p18ink4c expression. Tax binding to series of CDKs at specific points of the cell cycle (Pines, p15ink4b inactivated its function and made cells resis- 1993; Sherr, 1994). The activities of CDKs are positively tant to p15ink4b-mediated growth arrest induced by ␤ regulated by cyclins and negatively regulated by a family TGF . On the other hand, Tax did not bind to p18ink4c of CDK inhibitors including the INK4 family (Grana and and p19ink4d, but repressed transcription of p18ink4c gene through the E-box element of the promoter. Identi- fication of the E-box as a responsible element for tran-

1 scriptional repression is consistent with recent reports To whom correspondence and reprint requests should be ad- ␤ dressed at present address: Banyu Tsukuba Research Institute, Okubo on Tax-mediated trans-repression of DNA polymerase 3, Tsukuba, Ibaraki 300-2611, Japan. Fax: 81-298-77-2034. E-mail: and the lck and bax genes (Brauweiler et al., 1997; [email protected]. Lemasson et al., 1997; Uittenbogaard et al., 1994). The

suppression of the function and expression of INK4 family members possibly contributes to cell cycle deregulation in HTLV-1-infected cells.

RESULTS Interaction of Tax with INK4 family proteins Previously, we have demonstrated binding of Tax to p16ink4a protein (Suzuki et al., 1996), a member of the INK4 family of CDK inhibitors which contains four tandem repeats of the ankyrin motif (Serrano et al., 1993). Other members of the INK4 family, p15ink4b, p18ink4c, and p19ink4d, all contain similar ankyrin motifs to p16ink4a FIG. 2. Effect of p15ink4b and Tax on CDK4 kinase activity. Expres- and inhibit CDK4 activity (Grana and Reddy, 1995). Thus sion vectors for cyclin D1 (lanes 1–4), CDK4 (lanes 1–4), p15ink4b we were interested in whether Tax interacts with these (lanes 2–4), wild-type Tax (lane 3), and the d7/16 mutant (lane 4) were members of the INK4 family. We produced fusion pro- cotransfected into 293T cells. Total cell extracts were treated with anti-CDK4 antibody, and the immunoprecipitates were subjected to teins of INK4 family members with glutathione–S-trans- kinase assay by CDK4 using GST-pRb as the substrate followed by ferase (GST) and analyzed their binding to recombinant SDS–PAGE and autoradiography. Tax protein (His-Tax). The GST fusion proteins were ad- sorbed on glutathione–Sepharose beads and mixed with purified His-Tax. The complexes were collected by cen- action on CDK4 activity (Suzuki et al., 1996). To examine trifugation and subjected to Western blotting for detec- the similar effect of Tax on p15ink4b, the kinase activity tion of Tax protein. As shown in Fig. 1, Tax was detected of CDK4 was assayed in vitro using GST-pRb as a sub- in the complexes with GST-p15ink4b as well as GST- strate. Expression vectors for cyclin D1, CDK4, p15ink4b, p16ink4a (lanes 3 and 5), but not with GST alone (lane 1), and Tax were cotransfected into 293T cells and the cell GST-p18ink4c, or GST-p19ink4d (lanes 7 and 9). Tax mu- extracts were treated with anti-CDK4 antibody. The im- tant d7/16, which did not bind to p16ink4a, was not mune complexes were subjected to in vitro kinase as- detected in the complexes with p15ink4b, confirming says by adding GST-pRb and [␥32P]ATP, and the phos- binding specificity in vitro. phorylation of GST-pRb was analyzed by SDS–gel elec- trophoresis (Matsushime et al., 1994). When cyclin D1 Cancellation of p15ink4b-mediated inhibition of CDK4 and CDK4 were transfected, significant phosphorylation by Tax of GST-pRb was detected (Fig. 2, lane 1), but cotransfec- INK4 family proteins bind to cyclin D-dependent cata- tion of p15ink4b resulted in a decreased phosphorylation lytic subunit CDK4 or CDK6 and inhibit their kinase ac- (lane 2), consistent with the inhibitory effect of p15ink4b tivity (Grana and Reddy, 1995; Hunter and Pines, 1994). on CDK4 activity. Additional expression of Tax in this The binding of Tax to p16ink4a counteracts its inhibitory system restored the CDK4 kinase activity (lane 3), which had been suppressed by the expression of p15ink4b. These results indicate that Tax counteracted the inhibitory effect of p15ink4b on CDK4 kinase activity, similarly to p16ink4a. The inactive Tax mutant d7/16, which could not bind to p15ink4b, did not show any effect (lane 4), suggesting that the effect was mediated through the binding of Tax to p15ink4b.

Inhibition of TGF␤-induced arrest of cell growth p15ink4b was originally identified as a CDK inhibitor induced by TGF␤ (Hannon and Beach, 1994), which ar- FIG. 1. Binding of Tax protein to INK4 family proteins in vitro. Five rests the cell cycle at G1 (Reynisdottir et al., 1995). To nanograms of purified His-Tax (lanes 1, 3, 5, 7, 9) or its mutant d7/16 demonstrate the effect of Tax on p15ink4b-induced (lanes 2, 4, 6, 8, 10) was mixed with glutathione–Sepharose beads growth arrest, the expression vector for Tax was trans- ␮ containing 2 g each of GST (lanes 1, 2), GST-p15ink4b (lanes 3, 4), fected with a puromycin resistance gene into mouse GST-p16ink4a (lanes 5, 6), GST-p18ink4c (lanes 7, 8), or GST-p19ink4d (lanes 9, 10) and incubated for8hat4°C. The bound Tax was collected BaF3 cell line, and cell clones expressing Tax protein by centrifugation and analyzed by Western blotting with anti-Tax anti- were isolated. Tax-expressing BaF3 cells and control body. BaF3 cells were treated with increasing concentrations 386 SUZUKI ET AL. of TGF␤ for 10 h. The proliferation of cells was monitored by incorporation of BrdU into newly synthesized DNA. Treatment of control cells with TGF␤ decreased BrdU incorporation, indicating that TGF␤ induced cell growth arrest (Fig. 3A). Similar treatment of a Tax-expressing cell clone, on the other hand, reduced BrdU incorporation only slightly (Fig. 3A). This result indicates that Tax- expressing cells were resistant to TGF␤. Another Tax- expressing cell clone also gave similar results (data not shown), confirming that these observations were general to the cell clones expressing Tax. Enhanced expression of p15ink4b after TGF␤ treatment was observed at the mRNA level in Tax-expressing BaF3 cells, similarly to control cells (data not shown). In these Tax-expressing FIG. 4. Expression of p18ink4c and p19ink4d mRNA in various cell lines. Total RNA was isolated from various T-cell lines and analyzed by cells, Tax/p15ink4b complex was demonstrated by co- Northern blotting using 32P-labeled p18ink4c (A) or p19ink4d probe (B). immunoprecipitation after treatment with TGF␤ (Fig. 3B). The effects of Tax on expression of p18ink4c (C) and p19ink4d (D) were These results suggest that inactivation of p15ink4b by analyzed in Jurkat T-cells by infection with recombinant adenovirus Tax binding is the mechanism by which cells become vector expressing Tax (lane 10) or vector alone (lane 9). Cells were resistant to growth arrest induced by TGF␤. harvested 36 h after infection, and RNA preparations were analyzed by Northern blotting as in (A) and (B).

Down-regulation of p18ink4c by Tax As shown in Fig. 1, other members of the INK4 family, fected T-cell lines, although a transcript of 2.3 kb was p18ink4c and p19ink4d, did not bind to Tax, although they detected in uninfected T-cell lines tested (Fig. 4A). Ex- have ankyrin motifs. To understand their possible roles in pression of p19ink4d mRNA of 1.2 kb was detected in all HTLV-1-infected T-cells, we examined the expression of cell lines, although the levels were significantly lower in p18ink4c and p19ink4d in various T-cell lines infected HTLV-1-infected T-cell lines than in uninfected T-cell and uninfected with HTLV-1. Northern blot analysis re- lines (Fig. 4B). These results are consistent with previous vealed no detectable mRNA of p18ink4c in HTLV-1-in- observations in HTLV-1-infected T-cell lines (Akagi et al., 1996). The decreased expression of p18ink4c and p19ink4d in HTLV-1-infected T-cell lines is not likely to be the result of gross alteration of these two loci, since Southern blot analysis did not show any alteration of the specific bands of p18ink4c and p19ink4d in HTLV-1- infected T-cell lines compared with uninfected T-cell lines (data not shown). To determine whether Tax is responsible for the decreased expression of these genes, we analyzed the effect of Tax expression by infection of a recombinant adenovirus vector (Kanegae et al., 1994; Miyake et al., 1996; Niwa et al., 1991). Jurkat T-cells were infected with Tax-expressing virus or control virus, and RNA prepared 36 h after infection was analyzed by Northern blotting. Infection with Tax-expressing virus induced a significant reduction of p18ink4c mRNA, but infection with control FIG. 3. Resistance of Tax-expressing cells to TGF␤ treatment. (A) virus did not (Fig. 4C). However, expression of p19ink4d Effect of Tax on incorporation of BrdU. Tax-expressing (closed circles) was not affected by Tax expression under the experi- and unmodified (open circles) BaF3 cells were treated with TGF␤ (0.4, mental conditions used (Fig. 4D). These results indicate 1.0, 2.5 ng/ml) for 10 h. Then the cells were labeled with BrdU for 5 h that transient expression of Tax induced repression of and fixed. Incorporation of BrdU into DNA was estimated using anti- p18ink4c mRNA expression. BrdU antibody conjugated with peroxidase, and color emission was quantified by a microplate reader. Each experiment was performed at least three times and typical results are shown. (B) Detection of Tax/ Transcriptional repression of p18ink4c gene by Tax p15ink4b complex in TGF␤-treated cells. Tax-expressing cells were incubated with (lane 2) or without (lane 1) 2 ng/ml TGF␤ for 8 h, and the To investigate the mechanism for the repression of cell extracts were immunoprecipitated with anti-Tax antibody followed p18ink4c mRNA by Tax, we cloned the 5Ј-flanking region by Western blotting with anti-p15ink4b antibody. of human p18ink4c gene and analyzed the 2.9-kb up- EFFECTS OF TAX ON INK4 FAMILY PROTEINS 387

FIG. 5. Analysis of elements responsible for Tax-induced trans-repression in the promoter region of p18ink4c gene. (A) Structure of the 5Ј-regulatory region of the human p18ink4c gene. Genomic constitution is illustrated for the expression of 2.3-kb mRNA. Several transcriptional initiation sites are shown by vertical arrowheads. The consensus binding sites for several transcription factors are also indicated in the promoter region. (B) Mutation analysis of p18ink4c promoter. Each promoter region with a deletion mutation or an E-box mutation was linked to the luciferase gene and their promoter activities were analyzed in Hela cells with or without Tax expression vector. Luciferase activity in the absence of Tax and its ratio to that in the presence of Tax are presented. Each experiment was performed at least three times and typical results are shown. stream region from the ATG initiation codon by DNA scriptional promoter activity and repressive activity spe- sequencing. As shown in Fig. 5A, the genomic arrange- cifically responding to Tax. ment of p18ink4c was constructed by comparison of the To find the region responsible for the repression by cDNA sequence for 2.3 kb mRNA of p18ink4c (Blais et al., Tax, serial deletions from the 5Ј end were introduced into 1998). An S1 nuclease protection assay revealed five the promoter region and the promoter activities of the initiation sites for transcription at a region located 1.7 kb mutants were measured (Fig. 5B). In the absence of Tax, upstream of the translational initiation codon (data not a deletion of the promoter sequence from Ϫ1150 to Ϫ120 shown). had little effect on luciferase expression in the cells, but To examine whether the cloned 5Ј-flanking region has further deletion resulted in a significant decrease of promoter activity, a BamHI–SacI fragment of 540 bp con- activity, indicating that minimal promoter activity exists taining transcriptional initiation sites was inserted up- between Ϫ120 and ϩ610 (Fig. 5B). When Tax was coex- stream of the luciferase gene, constructing the reporter pressed with these reporter plasmids, the constructs plasmid, p18-B/S-Luc (Fig. 5B). A plasmid, p18-S/B-Luc, were efficiently trans-repressed by Tax up to the deletion containing the same fragment with the inverse orienta- at Ϫ710 (Fig. 5B). However, deletion up to Ϫ510 caused tion was used as a negative control. Each reporter plas- loss of the response to Tax, although its promoter activity mid was transfected into HeLa cells and luciferase ac- was retained. These results indicate that the responsible tivity was measured. The p18-B/S-Luc reporter ex- element for Tax-mediated repression was located be- pressed luciferase activity 50-fold higher than that of the tween Ϫ710 and Ϫ510 of the p18ink4c gene. negative control, which was almost equal to the back- This region contained an E-box consensus sequence ground level (data not shown). The luciferase activity which has been reported to be a potential target of Tax expressed by p18-B/S-Luc was inhibited by Tax expres- trans-repression (Brauweiler et al., 1997; Lemasson et sion (Fig. 5B) in a dose-dependent fashion (data not al., 1997; Uittenbogaard et al., 1994). Therefore, we intro- shown); however, it was not inhibited by a Tax mutant, duced a mutation into the E-box element (CAGGTG to d7/16, which is inactive in transcriptional activation (data TGGGTG). The promoter sequence with the mutant E-box not shown). These results indicate that the 1.8-kb Bam- element was no longer trans-repressed by Tax (Fig. 5B). HI–SacI fragment contained elements required for tran- Therefore, we conclude that the E-box element is re- 388 SUZUKI ET AL. quired for transcriptional repression of p18ink4c gene by human T-lymphocytes (Erickson et al., 1998). Therefore, Tax. the results of this study suggest that inactivation of p15ink4b by Tax is implicated in T-cell malignancy, pos- DISCUSSION sibly through alteration of the senescence program. p15ink4b was originally described as a TGF␤-induc- In this study, we found that Tax abrogated regulation of ible gene and is involved in TGF␤-mediated G1 arrest cyclin-dependent kinases by INK4 family proteins (Hannon and Beach, 1994; Reynisdottir et al., 1995). Pre- through two distinct mechanisms. One is direct binding viously, it was reported that HTLV-1-infected T-cell clones of Tax to p15ink4b and inactivation of its function, simi- were resistant to growth inhibition by TGF␤, and this larly to p16ink4a reported previously (Low et al., 1997; resistance correlated with lack of prevention of pRb Suzuki et al., 1996). The binding of Tax to p15ink4b phosphorylation (Hollsberg et al., 1994). Our findings on counteracted its inhibitory activity, causing CDK4 kinase the interaction of Tax with p15ink4b suggest that func- activation, resulting in resistance to TGF␤-induced tional inactivation of p15ink4b by Tax may be the molec- growth arrest. The other mechanism is trans-repression ular basis for the previous observation. In fact, we also of transcription of p18ink4c, resulting in elimination of the confirmed the unchanged phosphorylation status of pRb regulatory system from Tax-expressing cells. The trans- even after TGF␤ treatment (data not shown). These re- repression was mediated through the E-box element in sults support that Tax contributes to abnormal cell cycle the promoter of p18ink4c gene. progression of HTLV-1-infected T-cells. INK4 family proteins p16ink4a, p15ink4b, p18ink4c, In contrast to direct binding of Tax to p16ink4a and and p19ink4d function as negative regulators of the p15ink4b, we found that transient expression of Tax sup- mammalian cell cycle by their inhibitory activities against pressed the expression of 2.3 kb mRNA of p18ink4c, the cyclin D/CDK complex (Grana and Reddy, 1995; which may contribute to the decrease of p18ink4c in Hunter and Pines, 1994). Inhibition of the cyclin D/CDK HTLV-1-infected T-cell lines. For the Tax-induced sup- complex prevents the phosphorylation of pRb, which pression of 2.3-kb mRNA expression, the E-box element thus blocks the release of active transcription factors of in the promoter was required. In other reports, several the E2F family required for gene expression during G1/S sizes of mRNAs in human tissues (Guan et al., 1994) and progression. Lack or inactivation of INK4 proteins would two distinct promoters operating in mouse cells were therefore inactivate pRb through phosphorylation by an described (Phelps et al., 1998). However, in this study, we activated cyclin D/CDK complex, which would finally in- observed 2.3-kb mRNA as the highly predominant form in duce progression of the cell cycle. In fact, human human T-cell lines (Fig. 4A). Therefore, our analysis of p16ink4a and p15ink4b gene loci are tandemly aligned the expression of 2.3-kb mRNA should be sufficient for within a 30-kb DNA segment on chromosome 9p21 and understanding the major effect of p18ink4c on T-cell were frequently deleted in immortalized cell lines and lines. The mechanism of the E-box-mediated repression primary tumor cells (Drexler, 1998; Kamb et al., 1994; of 2.3-kb mRNA is now under investigation; however, it Nobori et al., 1994). The functional inactivation of would be shared with Tax-induced repression of DNA p15ink4b and transcriptional repression of p18ink4c by polymerase ␤ and the bax and lck genes (Brauweiler et Tax might mimic these genetic alterations and play sig- al., 1997; Lemasson et al., 1997; Uittenbogaard et al., nificant roles in the deregulation of cell cycle control. 1994). Our preliminary data show that a reporter gene From the high incidence of mutation of p16ink4a but with the isolated E-box in combination with a heterolo- not p15ink4b in many types of tumors, it has been clearly gous promoter is trans-repressed by Tax (manuscript in demonstrated that p16ink4a is a tumor suppressor gene. preparation), indicating that the E-box alone is sufficient In addition, p16ink4a has been proposed to be a central for trans-repression. Identification of factors involved in regulator of replicative senescence in fibroblastic cells trans-repression is in progress. (Alcorta et al., 1996; Hara et al., 1996; Serrano et al., Tax showed little effect on p19ink4d expression, al- 1997), based on the apparent link between loss of though its expression was significantly lowered in HTLV- p16ink4a and cellular immortality. In addition to these 1-infected T-cell lines (Fig. 4B). The mechanism of re- findings, recent studies have demonstrated important duced expression of p19ink4d in HTLV-1-infected T-cells roles of p15ink4b in cell growth and transformation, es- has not yet been determined, but whatever the mecha- pecially in T-lymphocytes: Selective inactivation of nism might be, a decrease or inactivation of members of p15ink4b through hypermethylation of p15ink4b gene the INK4 family seems to play a critical role in cell cycle was found in acute lymphoblastic leukemia and mouse deregulation in HTLV-1-infected T-cells. primary T-cell lymphoma independent of p16ink4a alter- Among the INK4 family proteins whose biochemical ation (Herman et al., 1996; Malumbres et al., 1997). Fur- properties are almost indistinguishable, genetic alter- thermore, accumulation of p15ink4b protein as well as ations of p18ink4c and p19ink4d genes have been found p16ink4a was implicated in replicative senescence in in only a few cases of human tumors, indicating that their EFFECTS OF TAX ON INK4 FAMILY PROTEINS 389 role in transformation is still open to question. However, protein, pGEX-pRb (379-928) plasmid was used (Matsu- p18ink4c-deficient mice provided suggestive data for its shime et al., 1994). role in T-cells (Franklin et al., 1998). These mice devel- To construct a recombinant adenovirus vector, the oped normally but showed gigantism, and their spleen coding sequence of Tax or lacZ was blunt-ended and and thymus were disproportionately enlarged and hyper- ligated into the SwaI site of a cosmid, pAdex1CAwt, plastic. T- and B-lymphocytes developed normally, but donated by Dr. Izumu Saito, University of Tokyo (Kanegae both exhibited a higher proliferative rate upon mitogenic et al., 1994; Miyake et al., 1996; Niwa et al., 1991). The stimulation, suggesting an important role of p18ink4c, recombinant adenovirus AxCAIY-Tax was prepared ac- especially in the regulation of lymphocytes. Therefore, cording to a procedure previously described (Kanegae et decreased expression of p18ink4c by Tax may contribute al., 1994) and purified through a CsCl cushion. Exponen- to increased growth of HTLV-1-infected T-cells, possibly tially proliferating cells were used for the viral infection. increasing the risk of malignancy. After 3 h, 10 ml fresh medium was added and cells were In this study, we found a more expanded role of Tax in then cultured for 36 h. RNA was isolated from the cells the cell cycle down-regulating INK4 family proteins and used for Northern blot analysis. through two distinct mechanisms. Cell cycle progression The BamHI/SacI 1.8-kb fragment (from Ϫ1150 to ϩ610) at G1/S is negatively regulated by the INK4 family pro- of the 5Ј-flanking sequence of the p18ink4c gene was teins and other CDK inhibitors. These inhibitors would inserted into the pGL3-basic reporter plasmid (Promega) function redundantly or specifically responding to vari- containing the luciferase gene, generating p18-B/S-Luc. ous signals. The effects of Tax on these INK4 family p18-1040 (Ϫ1040 to ϩ610), p18-710 (Ϫ710 to ϩ610), p18- members would disrupt the major parts of these regula- 510 (Ϫ510 to ϩ610), p18-420 (Ϫ420 to ϩ610), p18-120 tory systems. Although other inhibitors remain unaf- (Ϫ120 to ϩ610), and p18ϩ40 (ϩ40 to ϩ610) were 5Ј- fected, the observed effects of Tax would be effective in deletion mutants of the BamHI/SacI 1.8-kb genomic frag- lowering the threshold for initiation of abnormal cell ment; p18-B/S-Luc was treated sequentially with exonu- growth of HTLV-1-infected cells. clease III and mung bean nuclease, followed by self- ligation. Deletion endpoints were determined by DNA sequencing. p18-B/S-mutE-Luc was a mutant of the E- MATERIALS AND METHODS box element in p18-B/S-Luc, which was disrupted by Cells, plasmids, and antibodies site-directed mutagenesis (Kunkel et al., 1987) using a mutated oligonucleotide, 5Ј-CTCGGCCTCGCACCCAC- MT-1, MT-2, HUT102, MT-4, and Hayai are human T-cell CAGATCCATG-3Ј (Ϫ539 to Ϫ565). Mutation of the E-box lines infected with HTLV-1. Jurkat, CEM, and Molt-4 are from CAGGTG to TGGGTG was confirmed by DNA se- human T-cell lines. These T-cell lines were maintained in quencing. RPMI 1640 supplemented with 10% fetal calf serum Anti-p15ink4b antibody was donated by Dr. Hitoshi (FCS). BaF3 is a mouse IL-3-dependent proB cell line and Matsushime, Nippon Roche Research Center. Anti-Tax was maintained in RPMI 1640 supplemented with 10% antibody was described previously (Suzuki et al., 1996). FCS and 10% culture supernatant of WEHI cells, which Anti-CDK4 antibody was obtained from Santa Cruz Bio- are a source of IL-3. 293T, an adenovirus-transformed tech. human embryonic kidney cell line carrying SV40 large T antigen, and HeLa cells were maintained in Dulbecco’s In vitro assay for Tax binding to INK4 proteins modified Eagle’s medium (DMEM) supplemented with Each pGEX derivative was introduced into Escherichia 10% FCS. coli and the production of GST-INK4 proteins was in- The cDNAs of human p15ink4b, p18ink4c, and duced by 1 mM IPTG. The cells were recovered by p19ink4d were donated by Dr. David Beach, Cold Spring centrifugation (5000 g for 5 min) at 4°C and lysed by Harbor Laboratory, Dr. Yue Xiong, University of North sonication in phosphate-buffered saline (PBS) containing Carolina, and Dr. Hiroshi Hirai, University of Tokyo, re- Triton X-100. Cleared lysates were mixed with purified spectively. Fragments containing the entire coding se- histidine-tagged Tax and incubated for8hat4°C. The quences of these cDNAs were isolated by polymerase GST fusion proteins were collected on glutathione– chain reaction and cloned into the pGEX vector to pro- Sepharose 4B (Pharmacia) and analyzed by Western duce a GST fusion protein. The expression plasmid for blotting with anti-Tax antibody. p15ink4b in mammalian cells was constructed by insert- ing the coding sequence into the XbaI/BamHI site of the Immunoprecipitation and kinase assay pCG vector (Suzuki et al., 1996). The expression vectors for mouse cyclin D1, CDK4, and the puromycin resistance The 293T cells were transfected with plasmids by the gene were described previously (Matsushime et al., standard calcium phosphate precipitation procedure. 1994; Suzuki et al., 1996). For the production of GST-pRb Preparation of cell extracts and immunoprecipitation 390 SUZUKI ET AL.

were essentially the same as described previously (Ma- containing 8 M urea next to a DNA sequence ladder of tsushime et al., 1994). The precipitates were suspended the probe. in 30 ␮l kinase buffer (50 mM HEPES, pH 7.5, 10 mM MgCl , 1 mM DTT) containing 0.2 ␮g GST-pRb fusion 2 ACKNOWLEDGMENTS protein as a substrate and 2.5 mM EGTA, 10 mM ␮ NaH2PO4,10 MNa3VO4, 20 mM ATP, and 10 mCi We thank Dr. David Beach, Cold Spring Harbor Laboratory, Dr. Yue [␥32P]ATP (NEN Dupont). After incubation for 30 min at Xiong, University of North Carolina, and Dr. Hiroshi Hirai, University of 30°C, the samples were separated in SDS–polyacrylam- Tokyo, for providing cDNAs of the INK4 family. We thank Dr. Hitoshi Matsushime, Nippon Roche Research Center, for providing anti- ide gels followed by autoradiography. p15ink4b antibody. We also thank Dr. Izumu Saito, University of Tokyo, and Dr. Junichi Miyazaki, Osaka University, for providing the adenovirus Assay for cell proliferation expression system. This work was supported in part by a special grant for Advanced Research on Cancer from the Ministry of Education, BaF3 cells were cotransfected with a pCG-Tax expres- Culture, Sports and Science of Japan. sion plasmid and puromycin-resistance gene by electro- poration. At 36 h after transfection, cells were diluted and REFERENCES cultured in the presence of 0.5 ␮g/ml puromycin (Sigma) for 2–3 weeks. The expression of Tax protein in cell Akagi, T., Ono, H., and Shimotohno, K. (1996). Expression of cell-cycle clones was examined by Western blotting with anti-Tax regulatory genes in HTLV-I infected T-cell lines: possible involvement antibody. A cell growth assay was performed using a Cell of Tax1 in the altered expression of cyclin D2, p18Ink4 and p21Waf1/ Proliferation ELISA kit (Boehringer Mannheim) according Cip1/Sdi1. Oncogene 12, 1645–1652. Alcorta, D. A., Xiong, Y., Phelps, D., Hannon, G., Beach, D., and Barrett, to the manufacturer’s procedure. Tax-expressing clones J. C. (1996). Involvement of the cyclin-dependent kinase inhibitor p16 and control clone were cultured in 96-well microtiter (INK4a) in replicative senescence of normal human fibroblasts. Proc. plates and treated with increasing concentrations of Natl. Acad. Sci. USA 93, 13742–13747. TGF␤ for 10 h. Then the cells were labeled with BrdU for Blais, A., Labrie, Y., Pouliot, F., Lachance, Y., and Labrie, C. (1998). 5 h and fixed with ethanol. Incorporation of BrdU was Structure of the gene encoding the human cyclin-dependent kinase inhibitor p18 and mutational analysis in breast cancer. Biochem. detected using anti-BrdU antibody conjugated with per- Biophys. Res. Commun. 247, 146–153. oxidase and the color emission was quantified by a Brauweiler, A., Garrus, J. E., Reed, J. C., and Nyborg, J. K. (1997). microplate reader. Each experiment was performed at Repression of bax gene expression by the HTLV-1 Tax protein: least three times and typical results are shown. Implications for suppression of apoptosis in virally infected cells. Virology 231, 135–140. Chan, F. K., Zhang, J., Cheng, L., Shapiro, D. N., and Winoto, A. (1995). Isolation and characterization of the promoter region Identification of human and mouse p19, a novel CDK4 and CDK6 of the p18ink4c gene inhibitor with homology to p16ink4. Mol. Cell Biol. 15, 2682–2688. ␭ Drexler, H. G. (1998). Review of alterations of the cyclin-dependent A EMBL3 genomic library of human peripheral blood kinase inhibitor INK4 family genes p15, p16, p18 and p19 in human leukocytes was purchased from Clontech. Two million leukemia-lymphoma cells. Leukemia 12, 845–859. phages were screened with a 32P-labeled probe contain- Erickson, S., Sangfelt, O., Heyman, M., Castro, J., Einhorn, S., and ing the coding region of p18ink4c cDNA. Five clones after Grander, D. (1998). Involvement of the Ink4 proteins p16 and p15 in the second screening were amplified, purified, and ana- T-lymphocyte senescence. Oncogene 17, 595–602. Franklin, D. S., Godfrey, V. L., Lee, H., Kovalev, G. I., Schoonhoven, R., lyzed by restriction enzyme mapping with BamHI, EcoRI, Chen-Kiang, S., Su, L., and Xiong, Y. (1998). CDK inhibitors p18(INK4c) and SacI. All clones contained the 2.9-kb 5Ј-upstream and p27(Kip1) mediate two separate pathways to collaboratively region from the ATG initiation codon of human p18ink4c suppress pituitary tumorigenesis. Genes Dev. 12, 2899–2911. gene. The genomic structure of this region was deter- Grana, X., and Reddy, E. P. (1995). Cell cycle control in mammalian cells: mined by DNA sequencing, Northern blot analysis, and Role of cyclins, cyclin dependent kinases (CDKs), growth suppressor genes and cyclin-dependent kinase inhibitors (CKIs). Oncogene 11, comparison with cDNA sequences (Blais et al., 1998). 211–219. To determine the transcription start site, S1 nuclease Guan, K. L., Jenkins, C. W., Li, Y., Nichols, M. A., Wu, X., O’Keefe, C. L., protection mapping was performed. A 0.6-kb probe con- Matera, A. G., and Xiong, Y. (1994). Growth suppression by p18, a taining the region from Ϫ400 to ϩ200 of the 5Ј-flanking p16INK4/MTS1- and p14INK4B/MTS2-related CDK6 inhibitor, corre- region was synthesized using the Klenow fragment of lates with wild-type pRb function. Genes Dev. 8, 2939–2952. 32 Hannon, G. J., and Beach, D. (1994). p15INK4B is a potential effector of DNA polymerase I, P-labeled primer, single-stranded TGF-beta-induced cell cycle arrest. Nature 371, 257–261. DNA template, and restriction enzyme. DNA–RNA hybrid- Hara, E., Smith, R., Parry, D., Tahara, H., Stone, S., and Peters, G. (1996). ization was carried out with 100 ng DNA probe (5 ϫ 10 4 Regulation of p16CDKN2 expression and its implications for cell cpm) and 4 ␮g poly(A)ϩ RNA at 40°C overnight in buffer immortalization and senescence. Mol. Cell Biol. 16, 859–867. containing 80% formamide, 40 mM PIPES (pH 6.4), 400 Herman, J. G., Jen, J., Merlo, A., and Baylin, S. B. (1996). Hypermethyl- ation-associated inactivation indicates a tumor suppressor role for mM NaCl, and 1 mM EDTA. The hybrids were digested p15INK4B. Cancer Res. 56, 722–727. with 300 to 2000 units/ml S1 nuclease at 16°C for 30 min. Hinuma, Y., Nagata, K., Hanaoka, M., Nakai, M., Matsumoto, T., Ki- The products were analyzed on 4% polyacrylamide gels noshita, K. I., Shirakawa, S., and Miyoshi, I. (1981). Adult T-cell EFFECTS OF TAX ON INK4 FAMILY PROTEINS 391

leukemia: Antigen in an ATL cell line and detection of antibodies to Niwa, H., Yamamura, K., and Miyazaki, J. (1991). Efficient selection for the antigen in human sera. Proc. Natl. Acad. Sci. USA 78, 6476–6480. high-expression transfectants with a novel eukaryotic vector. Gene Hirai, H., Roussel, M. F., Kato, J. Y., Ashmun, R. A., and Sherr, C. J. (1995). 108, 193–199. Novel INK4 proteins, p19 and p18, are specific inhibitors of the cyclin Nobori, T., Miura, K., Wu, D. J., Lois, A., Takabayashi, K., and Carson, D-dependent kinases CDK4 and CDK6. Mol. Cell Biol. 15, 2672–2681. D. A. (1994). Deletions of the cyclin-dependent kinase-4 inhibitor Hollsberg, P., Ausubel, L. J., and Hafler, D. A. (1994). Human T cell gene in multiple human cancers. Nature 368, 753–756. lymphotropic virus type I-induced T cell activation. Resistance to Phelps, D. E., Hsiao, K. M., Li, Y., Hu, N., Franklin, D. S., Westphal, E., TGF-beta 1-induced suppression. J. Immunol. 153, 566–573. Lee, E. Y., and Xiong, Y. (1998). Coupled transcriptional and transla- Hunter, T., and Pines, J. (1994). Cyclins and cancer. II. Cyclin D and CDK tional control of cyclin-dependent kinase inhibitor p18INK4c expres- inhibitors come of age. Cell 79, 573–582. sion during myogenesis. Mol. Cell Biol. 18, 2334–2343. Inoue, J., Seiki, M., Taniguchi, T., Tsuru, S., and Yoshida, M. (1986). Pines, J. (1993). Cyclins and cyclin-dependent kinases: Take your part- Induction of interleukin 2 receptor gene expression by p40x encoded ners. Trends Biochem. Sci. 18, 195–197. by human T-cell leukemia virus type 1. EMBO J. 5, 2883–2888. Poeisz, B. J., Ruscetti, R. W., Gazdar, A. F., Bunn, P. A., Minna, J. D., and Jeang, K. T., Widen, S. G., Semmes, O. J. T., and Wilson, S. H. (1990). Gallo, R. C. (1980). Detection and isolation of the C retrovirus from HTLV-1 trans-activator protein, tax, is a trans-repressor of the human fresh and cultured lymphocytes of a patient with T cell lymphoma. beta-polymerase gene. Science 247, 1082–1084. Proc. Natl. Acad. Sci. USA 77, 7415–7419. Kamb, A., Gruis, N. A., Weaver-Feldhaus, J., Liu, Q., Harshman, K., Reynisdottir, I., Polyak, K., Iavarone, A., and Massague, J. (1995). Kip/Cip Tavtigian, S. V., Stockert, E., Day, R. S. R., Johnson, B. E., and Skolnick, and Ink4 Cdk inhibitors cooperate to induce cell cycle arrest in M. H. (1994). A cell cycle regulator potentially involved in genesis of response to TGF-beta. Genes Dev. 9, 1831–1845. many tumor types. Science 264, 436–440. Serrano, M., Hannon, G. J., and Beach, D. (1993). A new regulatory motif Kanegae, Y., Makimura, M., and Saito, I. (1994). A simple and efficient in cell-cycle control causing specific inhibition of cyclin D/CDK4. method for purification of infectious recombinant adenovirus. Jpn. Nature 366, 704–707. J. Med. Sci. Biol. 47, 157–166. Serrano, M., Lin, A. W., McCurrach, M. E., Beach, D., and Lowe, S. W. Kunkel, T. A., Roberts, J. D., and Zakour, R. A. (1987). Rapid and efficient (1997). Oncogenic ras provokes premature cell senescence associ- site-specific mutagenesis without phenotypic selection. Methods ated with accumulation of p53 and p16INK4a. Cell 88, 593–602. Enzymol. 154, 367–382. Sherr, C. J. (1994). G1 progression: Cycling on cue. Cell 79, 1059–1065. Lemasson, I., Robert-Hebmann, V., Hamaia, S., Duc Dodon, M., Gazzolo, Suzuki, T., Kitao, S., Matsushime, H., and Yoshida, M. (1996). HTLV-1 Tax L., and Devaux, C. (1997). Transrepression of lck gene expression by human T-cell leukemia virus type 1-encoded p40tax. J. Virol. 71, protein interacts with cyclin-dependent kinase inhibitor p16INK4A 1975–1983. and counteracts its inhibitory activity towards CDK4. EMBO J. 15, Low, K. G., Dorner, L. F., Fernando, D. B., Grossman, J., Jeang, K. T., and 1607–1614. Comb, M. J. (1997). Human T-cell leukemia virus type 1 Tax releases Uchiyama, T., Yodoi, J., Sagawa, K., Takatsuki, K., and Uchino, H. (1977). cell cycle arrest induced by p16INK4a. J. Virol. 71, 1956–1962. Adult T-cell leukemia: Clinical and hematologic features of 16 cases. Malumbres, M., Perez de Castro, I., Santos, J., Melendez, B., Mangues, Blood 50, 481–492. R., Serrano, M., Pellicer, A., and Fernandez-Piqueras, J. (1997). Inac- Uittenbogaard, M. N., Armstrong, A. P., Chiaramello, A., and Nyborg, tivation of the cyclin-dependent kinase inhibitor p15INK4b by dele- J. K. (1994). Human T-cell leukemia virus type I Tax protein represses tion and de novo methylation with independence of p16INK4a alter- gene expression through the basic helix-loop-helix family of tran- ations in murine primary T-cell lymphomas. Oncogene 14, 1361– scription factors. J. Biol Chem. 269, 22466–22469. 1370. Yoshida, M. (1993). HTLV-1 Tax: Regulation of gene expression and Matsushime, H., Quelle, D. E., Shurtleff, S. A., Shibuya, M., Sherr, C. J., disease. Trends Microbiol. 1, 131–135. and Kato, J.-Y. (1994). D-type cyclin-dependent kinase activity in Yoshida, M. (1995). HTLV-1 oncoprotein Tax deregulates transcription of mammalian cells. Mol. Cell Biol. 14, 2066–2076. cellular genes through multiple mechanisms. J. Cancer Res. Clin. Miyake, S., Makimura, M., Kanegae, Y., Harada, S., Sato, Y., Takamori, Oncol. 121, 521–528. K., Tokuda, C., and Saito, I. (1996). Efficient generation of recombinant Yoshida, M., Miyoshi, I., and Hinuma, Y. (1982). Isolation and charac- adenoviruses using adenovirus DNA-terminal protein complex and a terization of retrovirus from cell lines of human adult T-cell leukemia cosmid bearing the full-length virus genome. Proc. Natl. Acad. Sci. and its implication in the disease. Proc. Natl. Acad. Sci. USA 79, USA 93, 1320–1324. 2031–2035.